An inexpensive and rapid diagnostic method of Koi Herpesvirus (KHV) infection by loop-mediated isothermal amplification

BACKGROUND: Koi Herpesvirus (KHV) affects both juvenile and adult common carp and koi, and is especially lethal to fry. The high mortalities caused by the disease have had a negative impact on the international koi trade. Different diagnostic techniques have been used to detect KHV, including: isolation of the virus in cell culture, electron microscopy, several PCR tests, ELISA and in situ hybridisation. All of these methods are time consuming, laborious and require specialised equipment. RESULTS: A rapid field diagnosis of KHV in common and koi carp was developed using loop-mediated isothermal amplification (LAMP). The LAMP reaction rapidly amplified nucleic acid with high specificity and efficiency under isothermal conditions using a simple water bath. Two methods of extracting DNA from host tissue were compared: extraction by boiling and by using a commercial extraction kit. A set of six primers – two inner primers, two outer primers and two loop primers – was designed from a KHV amplicon. The reaction conditions were optimised for detection of KHV in 60 min at 65°C using Bst (Bacillus stearothermophilus) DNA polymerase. When visualised by gel electrophoresis, the products of the KHV LAMP assay appeared as a ladder pattern, with many bands of different sizes from 50 base-pairs (bp) up to the loading well. The KHV LAMP product could also be simply detected visually by adding SYBR Green I to the reaction tube and observing a colour change from orange to green. All samples positive for KHV by visual detection were confirmed positive by gel electrophoresis. The KHV LAMP had the same sensitivity as a standard PCR assay for the detection of KHV. CONCLUSION: This paper describes an accelerated LAMP assay for diagnosis of KHV. The entire procedure took only 90 minutes to produce a result: 15 minutes for DNA extraction; 60 min for the LAMP reaction; 2 min for visual detection using SYBR Green I. The test can be used under field conditions because the only equipment it requires is a water bath

Loop-mediated isothermal amplification as an emerging technology for detection of Yersinia ruckeri the causative agent of enteric red mouth disease in fish

<p>Abstract</p> <p>Background</p> <p>Enteric Redmouth (ERM) disease also known as Yersiniosis is a contagious disease affecting salmonids, mainly rainbow trout. The causative agent is the gram-negative bacterium <it>Yersinia ruckeri</it>. The disease can be diagnosed by isolation and identification of the causative agent, or detection of the <it>Pathogen </it>using fluorescent antibody tests, ELISA and PCR assays. These diagnostic methods are laborious, time consuming and need well trained personnel.</p> <p>Results</p> <p>A loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for detection of <it>Y. ruckeri </it>the etiological agent of enteric red mouth (ERM) disease in salmonids. The assay was optimised to amplify the <it>yruI/yruR </it>gene, which encodes <it>Y. ruckeri </it>quorum sensing system, in the presence of a specific primer set and <it>Bst </it>DNA polymerase at an isothermal temperature of 63°C for one hour. Amplification products were detected by visual inspection, agarose gel electrophoresis and by real-time monitoring of turbidity resulted by formation of LAMP amplicons. Digestion with <it>Hph</it>I restriction enzyme demonstrated that the amplified product was unique. The specificity of the assay was verified by the absence of amplification products when tested against related bacteria. The assay had 10-fold higher sensitivity compared with conventional PCR and successfully detected <it>Y. ruckeri </it>not only in pure bacterial culture but also in tissue homogenates of infected fish.</p> <p>Conclusion</p> <p>The ERM-LAMP assay represents a practical alternative to the microbiological approach for rapid, sensitive and specific detection of <it>Y. ruckeri </it>in fish farms. The assay is carried out in one hour and needs only a heating block or water bath as laboratory furniture. The advantages of the ERM-LAMP assay make it a promising tool for molecular detection of enteric red mouth disease in fish farms.</p

Delivering the pain:an overview of the type III secretion system with special consideration for aquatic pathogens

Gram-negative bacteria are known to subvert eukaryotic cell physiological mechanisms using a wide array of virulence factors, among which the type three-secretion system (T3SS) is often one of the most important. The T3SS constitutes a needle-like apparatus that the bacterium uses to inject a diverse set of effector proteins directly into the cytoplasm of the host cells where they can hamper the host cellular machinery for a variety of purposes. While the structure of the T3SS is somewhat conserved and well described, effector proteins are much more diverse and specific for each pathogen. The T3SS can remodel the cytoskeleton integrity to promote intracellular invasion, as well as silence specific eukaryotic cell signals, notably to hinder or elude the immune response and cause apoptosis. This is also the case in aquatic bacterial pathogens where the T3SS can often play a central role in the establishment of disease, although it remains understudied in several species of important fish pathogens, notably in Yersinia ruckeri. In the present review, we summarise what is known of the T3SS, with a special focus on aquatic pathogens and suggest some possible avenues for research including the potential to target the T3SS for the development of new anti-virulence drugs. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13567-021-01015-8

Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta

Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan

Vertical transmission of Tetracapsuloides bryosalmonae (Myxozoa), the causative agent of salmonid proliferative kidney disease

license: Copyright © Cambridge University Press 2013 0000-0001-7279-715Xlicense: Copyright © Cambridge University Press 2013. The attached document is the authors' final accepted version of the journal article. You are advised to consult the publisher's version if you wish to cite from it

To the very End. A contrastive study of N-Rhemes in English and Swedish translations

The present study is an explorative, corpus-based contrastive study of N-Rhemes in English and Swedish original texts, as well as translations between the two languages. The aim is twofold, to describe the N-Rheme in English and Swedish Fiction and Popular Science texts, and to examine translation correspondences, and lack of correspondences, between English and Swedish N-Rhemes. The first part of the investigation shows that N-Rhemes are very similar in the two languages and the two text types. The main differences are to a great extent related to word order differences between the two languages, e.g. the V2-constraint in Swedish and Subject prominence in English. However, word order is not the only explanation. Frequently, there is interplay between word order and information structure, as Swedish is more backwards-oriented and seems to follow the principle of end-weight more strictly than English. The analysis of the translation (non)-correspondences: Full Match, Reformulation, Movement and Restructuring shows that more translation changes occur in the translations into English. Reformulations are most frequent and typically result in unit shifts, function shifts and explicitness changes. Furthermore, the results show that English and Swedish clearly have different clause structure preferences. English favours hypotactic structures where Swedish has paratactic structures, which is reflected in the translations. In the translations into Swedish, clauses are frequently split, resulting in T-units that are informationally less dense, whereas in the translations into English, clauses are merged, resulting in informationally denser clauses. When information density is increased or decreased, this frequently results in explicitness changes. Finally, many of the translation changes could be seen as related to the character of the N-Rheme. N-Rhemes are often long and complex, and present newsworthy information. The longer the N-Rheme, the more information that potentially could be changed in the translation process. The great number of Reformulations and Restructurings reflects how translation changes occur with a purpose to ascertain that the goals of the texts are preserved, and even made clearer in the translation